Project Summary The bipotential differentiation of liver progenitor cells is integral to liver development, regeneration, and diseases including bile duct paucity and liver cancer. The regionalization of biliary differentiation and morphogenesis near the portal region of the liver has suggested that spatially segregated microenvironmental signals govern this process. Accordingly, both TGF? and Notch signaling have been demonstrated to be activated in the portal area, and both have been shown to be critical mediators of bile duct formation. We have recently developed a cell microarray platform that enabled the systematic analysis of liver progenitor fate specification within defined microenvironments, and in particular, facilitated the assessment of interactions between TGF?, Notch, and other microenvironmental cues including extracellular matrix. Utilizing this approach, we additionally observed a reproducible gradient of biliary differentiation within the multicellular patterns of the array. Building on these findings, we hypothesize that mechanical signals play a role in progenitor function, and that spatial patterns of cell mechanotransduction and Notch signaling act together to regulate differentiation fate. We will exploit the tightly controlled and tunable microenvironments of the cell microarray platform to investigate the influence of multicellular geometry, cell-cell and cell-substrate interactions, and interrelated cell mechanical stress in biliary versus hepatocytic differentiation. Further, we will examine crosstalk between mechanical cues and Notch signaling, including the potential involvement of Hippo pathway effectors in these signaling interactions. We anticipate that these studies will reveal new insights into the combinatorial regulation of liver progenitor fate and will also establish this platform as a tool for studying disease mechanisms.